The document discusses fixation and processing of tissue specimens. It describes fixation as a process that preserves cells and tissues in a physical and chemical state to prevent biochemical changes and morphological distortion. The goals of fixation are to maintain the original tissue structure and prevent autolysis and bacterial growth. Common fixatives include formaldehyde and ethanol. The document outlines various types of fixatives and factors that influence fixation like buffer, pH, duration, temperature and concentration. It also discusses processing of fixed tissue, which provides rigidity for sectioning. Processing is influenced by viscosity, agitation, heat, vacuum and pressure. The document notes potential artifacts from improper fixation like formalin pigment or prolonged fixation effects.

1. FIXATION AND PROCESSING
OF TISSUE SPECIMEN
Dr. Atreyee Chakrabarty
Junior Resident-1 (MD Pathology)
Institute of Medical Sciences, Banaras Hindu University
Moderators: Dr Bitan Naik and Dr Vishal Kumar

2. FIXATION

3. WHAT IS FIXATION?
 Process - cells and tissues are fixed in a physical
and chemical state
 Prevents biochemical or proteolytic activities in
the cell.
 Resist morphological change or distortion or
decomposition
 Fixation maintains the original microscopic
relationship between cells, cellular components
and the extracellular material

4. WHY DO WE NEED TO FIX A
TISSUE?
 Autolysis is the postmortem degeneration of cells
with release of their contents into the interstitial
space, where they may provoke inflammation
 Putrefaction - decomposition of cells by bacteria
and other organisms that invade the tissue, to release
by products.

5. AIMS OF FIXATION
 To preserve the tissue in the state nearest to its
original living state
 To prevent any change in shape or size of the
tissue at the time of processing
 To prevent autolysis
 To prevent bacterial growth
 To have a clear stain
 To have better optical quality of the cells.

6. IDEAL FIXATIVE
 Should maintain the original
shape and size and recovery
of macromolecules
 Should be usable for a wide
variety of tissue types
 Should prevent autolysis and
prevent bacterial
decomposition
 Should provide a consistently
high quality stain
 Should penetrate
tissues rapidly
 Should have a good
shelf life
 Should be disposable
and recyclable, cost
effective.
 Should have good
toxicological and
flammability profile
to permit safe use.

7. Till date, an ideal fixative has not been identified.
So we choose a fixative based on requirement.
Histopathology:10% neutral buffered formalin
Cytology:95% ethyl alcohol.

8. CHANGES IN A TISSUE DURING
FIXATION
1. Volume changes
2. Tissue hardening
3. Interference with staining
4. Change in optical density

9. VOLUME CHANGES
 Fixatives - swelling or shrinkage
 Formaldehyde causes 30% reduction in volume.
 Bahr et al. noted that:
Formaldehyde
concentration
Degree of tissue shrinkage
1

10.  Glutaraldehyde also causes shrinkage. But with
osmium tetroxide, 70% increase in cell size.
 Osmium tetroxide causes cellular swelling by
three mechanisms:
1. Altered membrane permeability
2. Inhibition of the respiratory enzymes
3. Change in Na+ ion transport

11. TISSUE HARDENING
A change in the consistency of the tissue after
applying fixative

12. INTERFERENCE WITH STAINING
 Fixation may cause hindrance to staining.
 Osmium tetroxide inhibits hematoxylin and eosin
staining.
 Hence, after staining with H and E in osmium
tetroxide fixed tissues, poor tissue differentiation
and staining artefacts are seen.

13. CHANGE IN OPTICAL DENSITY
Change in optical density of the nuclei - nuclei may
appear condensed and hyperchromatic.

14. TYPES OF FIXATIVES
Types of
fixatives
Physical
Heat fixation
Microwave
fixation
Freeze drying
and freeze
substitution
Chemical
Coagulative Cross linking

15. PHYSICAL FIXATIVES

16. HEAT FIXATION
 Simplest form of fixation
 Adequate morphology can be obtained by
boiling the tissue in normal saline
 Used to accelerate the other forms of
fixation as well as the other steps of
tissue processing.

17. MICROWAVE FIXATION
Microwaving reduces the
time of fixation.
Microwave generates
electromagnetic field
Dipolar molecules like
water oscillate rapidly
Kinetic energy
Heat generation
Uniform increase in
temperature of the tissue

18.  However, microwaving tissue in formalin can
produce toxic and potentially explosive vapours.
 Solutions to prevent this:
1. Hood for extraction of these vapours
2. Using commercial glycol based fixatives at 55°C

19. FREEZE DRYING
The
tissue is
cut into
thin
sections
Immersed
in a liquid
coolant at
-160 °C.
The
tissue is
placed in
the
vacuum
chamber
at -30 to
-50 °C.
The
tissue is
gradually
warmed
to 4 °C
Embedded

20. FREEZE SUBSTITUTION
 Similar to freeze drying
 Specimen is immersed in a liquid fixative like
acetone at -40°C
 Used in research environment, rarely used in
clinical laboratory settings.

21. CHEMICAL FIXATIVES

22. COAGULANT FIXATIVES
 Cellular architecture in vivo is primarily
maintained by lipoproteins and fibrous proteins
such as collagen.
 So, coagulating these proteins maintains tissue
histomorphology at light microscopic level.

23.  Coagulant fixatives result in cytoplasmic
flocculation and poor preservation of
mitochondria and secretory granules.
 So, it is not used in ultrastructural analysis.
COAGULANT
FIXATIVES
DEHYDRATING OTHER TYPES

24. DEHYDRATING COAGULANT
FIXATIVES
 Methanol, ethanol and acetone.
 Act by destabilizing the hydrophilic and
hydrophobic interactions within the proteins.
 Together, these changes disrupt the tertiary
structure of the proteins.
 This changes their physical properties and
causes loss of function.

25.  Denaturation of proteins by alcohol depends on:
1. Choice and concentration of alcohol
2. Presence of organic and inorganic substances
3. pH of fixation
4. Temperature of fixation
 Protein denaturing effect of different
coagulant fixatives:
Ethanol>Phenol>Water and polyhydric
alcohol>monocarboxylic acid>dicarboxylic acid

26. OTHER TYPES OF COAGULANT
FIXATIVES
 Acetic acid, Trichloroacetic acid and Picric acid.
 Charges on the ionisable side chains are changed
using acid coagulants by disrupting the
electrostatic and hydrogen bonding.
 These acids can also insert a lipophilic anion into a
hydrophilic region and disrupt the tertiary
structure of proteins.

27. ACETIC ACID TRICHLOROACETIC
ACID
PICRIC ACID
It coagulates the
nucleic acids but does
not fix or precipitate
proteins. So, it is used
with other fixatives to
prevent loss of nucleic
acids.
Penetrates the
hydrophobic domains
of proteins and the
anion produced reacts
with charged amine
groups.
This reaction
precipitates the
proteins and extracts
the nucleic acid.
Picric acid fixation
produces brighter
staining but low pH
solution may cause
hydrolysis and loss of
nucleic acids.

28. CROSS LINKING FIXATIVES
 Also known as “covalent additive fixatives”
 They form cross links within and between proteins
and nucleic acids
 Examples:
1. Formaldehyde
2. Glutaraldehyde
3. Other aldehydes like chloral hydrate and glyoxal
4. Metal salts like mercuric and zinc chloride
5. Metallic compounds such as osmium tetroxide

29. FACTORS AFFECTING FIXATION

30. BUFFER AND PH
- Effect depends on application of formalin
- In strongly acidic environment, formalin may affect
protein structure and forms formalin pigment.
- Common buffers include phosphate and bicarbonate.
- Unbuffered formalin is better than neutral buffered
formalin for immunorecognition of antigens.
- In most cases, neutral buffered formalin is preferred.

31. DURATION OF FIXATION AND SIZE
OF SPECIMEN
The duration of fixation is given by the following
formula:
d = k √t
d=depth needed to reach by a fixative (in mm)
k=Constant of diffusibility, fixative specific
t= time taken (in hours)
 Formalin penetrates at the rate of 1 mm/hr
 Rapid fixation is acceptable as long as histochemical
staining remains adequate.

32. TEMPERATURE OF FIXATION
 The diffusion of molecules increases with rising
temperature due to their more rapid movement
and vibration.
 The rate of penetration of a tissue by
formaldehyde is faster at higher temperatures.

33. CONCENTRATION OF FIXATIVE
 Concentrations of formalin above 10% tend to
cause increased tissue hardening.
 Ethanol concentrations below 70% do not remove
free water from tissues efficiently.

34. OSMOLALITY OF FIXATIVES AND
IONIC COMPOSITION
 Hypertonic and hypotonic solutions lead to
shrinkage and swelling, respectively.
 The best morphological results are obtained with
solutions that are slightly hypertonic (400–450
mOsm),
 However, osmolality for 10% NBF is about 1500
mOsm.
 Similarly, various ions (Na+, K+, Ca2+, Mg2+) can
affect cell shape and structure regardless of the
osmotic effect.

35. FORMALDEHYDE
- Most commonly used fixative
- Pure formaldehyde is a vapour
- Formalin= Aqueous solution of formaldehyde
- Formalin contains 37-40% formaldehyde
- Commonly used: 10% solution of formalin- contains
4% weight to volume of formaldehyde.

36. MECHANISM OF ACTION OF FORMALIN
Once these bridges have
formed, formalin cannot be
removed from the tissue by
simple washing.

37. ADVANTAGES:
1. High penetration rate
2. Cell morphology is preserved well in formalin
3. Solution is easy to make
4. Inexpensive
DISADVANTAGES:
1. Slow fixation
2. Fails to preserve acid mucopolysaccharides
3. Can form artefacts
4. Can affect skin causing dermatitis and chronic
inhalation can cause bronchitis.

38. DIFFERENT SOLUTIONS OF
FORMALIN
 Neutral buffered formalin: Most commonly used
 Carson’s modified Millonig’s phosphate buffered
formalin: better at ultrastructural preservation than
neutral buffered formalin
 Formalin calcium acetate: good for preservation of
lipids
 Formal (10% formalin) buffered saline
 Formal (10% formalin) zinc, unbuffered: Excellent for
immunohistochemistry
Tap water 900 ml
Formaldehyde
(37%)
100 ml
Sodium
phosphate,
monobasic
monohydrate
4 g
Sodium
phosphate,
dibasic,
anhydrous
6.5 g
COMPOSITION OF 10%
NEUTRAL BUFFERED
FORMALIN

39. MERCURIC FIXATIVES
 Zenker’s solution: Good fixative for bloody specimens
and trichrome stains
 Helly’s solution: Excellent for bone marrow
extramedullary hematopoiesis and intercalated discs.
 Schnaudinn’s solution
 Ohlmacher’s solution
 Carnoy Lebraun solution
 B5 fixative: Frequently used for bone marrow, lymph
nodes, spleen and other hematopoietic tissues

40. DICHROMATE FIXATIVES
1. Miller’s solution
2. Moller’s or Regaud’s fluid
3. Orth’s solution

41. PICRIC ACID FIXATIVES
1. Bouin’s solution: fixation of lymph nodes, prostate
and kidney biopsies.
2. Hollande’s solution: useful for GI biopsies and
endocrine tissues.

42. OTHER DEHYDRANT FIXATIVES
1. Clarke’s solution
2. Carnoy’s fixative:
 useful for RNA stains and glycogen preservation.
 may destroy staining of acid fast bacilli.
 Also useful in cytology to clear heavily blood
stained specimens.
3. Methacarn’s fixative:
Causes less shrinking and less hardening than
Carnoy, but pattern of staining is the same.

43. DEHYDRANT CROSS LINKING
FIXATIVES
1. Carson’s formula
2. Alcoholic formalin
3. Alcohol-formalin-acetic acid fixative
4. Gendre’s solution
5. Aged alcoholic Bouin equivalent
6. Rossman’s solution

44. FIXATIVES IN CYTOLOGY
 Rapid fixation of smears- to preserve cytological
details of cells spread on a glass slide.
 Marked cellular distortion if smears are air dried
before fixation.
 Previous fixative of choice: Ether and 95% ethanol
 Current fixative of choice: 95% ethanol

45.  Smears should be kept in this solution for 15 mins
prior to staining.
 EQUIVALENT CONCENTRATIONS OF SEVERAL
ALCOHOLS FOR PURPOSES OF CELL FIXATION:
1. 100% Methanol
2. 95% Ethanol
3. 95% Denatured alcohol
4. 80% Propanol
5. 80% Isopropanol

46. COATING FIXATION
 Can be sprayed or applied with a dropper to freshly
prepared smears,
 Eliminates the use of bottles and fixing solutions.
 Have a dual action: fix the cells and form a thin,
protective coating over the smear.
 Not recommended for smears prepared from fluids
within the laboratory.

47.  Should be applied immediately to fresh smears.
 Danos-Holmquist tested several spray fixatives
and found that the distance of 10-12 inches
between the nozzle and the smear was optimal.
 Two commonly used spraying fixatives are:
1. Polyethylene glycol
2. Diaphane fixative

48. PERFUSION FIXATION
 Mainly used for research purposes
 The fixative solution is infused in the arterial
system of the animal and the whole animal is
fixed.
 Some organs like brain and spinal cord can also be
fixed by perfusion fixation

51. PRECAUTIONS TO BE TAKEN FOR
PROPER FIXATION
 Gross specimens should not rest on the bottom of the
container of a fixative.
 Should be separated from the bottom by a wadded
fixative soaked paper or cloth to allow penetration of
the fixative from all directions.
 Unfixed gross specimens which are to be cut and stored
in fixative prior to processing should not be thicker than
5 mm.

52.  Gross specimens kept in blood or bloody fluids
should be thoroughly washed with saline before
putting into the fixative.
 The fixative volume should be at least 10 times
the volume of the tissue specimen for rapid
fixation.

53. FIXATION
ARTEFACTS
FORMALIN
PIGMENT
MERCURY
PIGMENT
FUZZY
STAINING
PROLONGED
FIXATION
ARTEFACTS
DICHROMATE
DEPOSITS
- Insoluble
brownish black
refractile
birefringent
extracellular
pigment due to
reaction of
formalin with
hemoglobin
derivatives.
- Can be avoided by
using neutral
formalin
- Dark brown
irregular
deposits
- Can be
removed by
using iodine
and sodium
thiosulphate
- Nuclear and
cytoplasmic
details are
obscured
- Section
looks fuzzy
or hazy
- Due to
insufficient
fixative
amount or
time.
- Prolonged
fixation can
cause
shrinkage of
tissue
followed by
separation
- The tissue
may show
holes or
empty spaces
within it
- If the tissue is
not properly
washed after
dichromate
fixation,
chromium salts
may form
resulting in an
artefact.

54. FORMALIN PIGMENT
ARTEFACT
MERCURY PIGMENT
ARTEFACT
FUZZY STAINING
PROLONGED
FIXATION
ARTEFACT

55. PROCESSING

56. WHAT IS PROCESSING?
 Next step after fixation
 Poor processing of tissue may affect section
cutting and staining.
 To provide sufficient rigidity to the tissue
 Allows tissues to be cut into thin sections for
microscopic examination

57. FACTORS INFLUENCING PROCESSING

58. VISCOSITY AGITATION HEAT VACCUM AND
PRESSURE
PROCESSING
SOLVENT
CONTAMINATI
ON
Low viscosity-
smaller sized
molecules and
faster penetration
rate.
-Increases the
flow of solutions
around the tissue.
-Mechanism is
vertical or
rotatory
oscilation or
pressurized
removal and
replacement of
fluids at time
intervals.
-Improves the
fluid exchange
and penetration
rate
-Ensure that there
are no heat
artefacts or
shrinkage or
hardening of the
tissue
Increase fluid
mobility,
infiltration rate
and decreases
time for each
step
-Vaccum aids the
removal of air
pockets in porous
tissue eg: lung
The number of
blocks on each
run, tissue type,
size, frequency of
the runs, use of
sponges and cross
contamination of
processor solvents
will influence how
often solutions
should be rotated
or changed to
maintain
processing
contamination

59. STAGES OF TISSUE PROCESSING
 DEHYDRATION: Removes water and unbound fixative from
the tissue
 CLEARING: Displaces dehydrating solutions, making the
tissue components receptive to the infiltrating medium
 INFILTRATION: Permeates tissue with a support medium
 EMBEDDING: Orientation of the tissue sample in a support
medium to create a tissue block suitable for sectioning

60. POST FIXATION TREATMENT
 Specimens fixed in alcohol fixatives should be
followed with alcohol to prevent reintroduction of
water into the tissue specimen.
 The alcohol concentration will depend on the alcohol
concentration of the fixative.
 Picric acid fixatives will colour the tissue bright
yellow. So the tissue should be rinsed in 50% alcohol
for 4-6 hours to remove the excess fixative.

61. DEHYDRATION
 Dehydration displaces the residual fixative as well
as the cellular water.
 Water is available in two forms: bound and free.
 The bound water is part of the integral
macromolecules.
 Graded alcohols are used to remove the free
water
 Exposure to heat or higher grade alcohols (95-
100%) removes the bound water.

62.  OVER DEHYDRATION: Produces “parched earth
effect” during microtomy.
 INCOMPLETE DEHYDRATION: Impairs the
penetration of clearing agents into the tissue and
leave the specimen soft and non receptive to
paraffin wax infiltration.
 COMMONLY USED REAGENTS:
1. Ethanol
2. Isopropanol
3. Glycol ether dehydrants

63. CLEARING
 To remove the dehydrating agent itself from the
tissue
 Many dehydrating agents are not miscible with the
impregnating material (paraffin wax).
 Clearing agents also make the tissue clear and
improve the microscopic examination

64.  Must be miscible with both the dehydrants and
paraffin wax.
 This ensures complete impregnation
 Selection of an appropriate clearing agent
depends on:
a. Type of tissue and processor
b. Processing conditions such as heat and vaccum
c. Safety factors and cost.

65.  VOLUME OF THE CLEARING AGENT: It should be
40 times the volume of the specimen.
 TOTAL DURATION OF CLEARING: 1 hour for
smaller tissues and three changes in toluene for
60 mins each in case of larger tissues
 END POINT DETECTION: The tissue will look
transparent
 COMMONLY USED CLEARING AGENTS:
1. Xylene: most common
2. Toluene
3. Amyl nitrate
4. Cedarwood oil

66. IDEAL CLEARING AGENT
1. Low viscosity and high penetration rate
2. Low melting point
3. Miscible with both alcohol and molten wax
4. No tissue damage
5. Less toxic and inflammable
6. Inexpensive

67. XYLENE
 It is an excellent lipid solvent.
 It removes water from the tissues. So, tissues may
become hard and brittle.
 Small pieces of tissue are rapidly cleared within
60 mins.
 However, xylene is inflammable and irritant but
less toxic than chloroform
 It is inexpensive.

68. TOLUENE CHLOROFORM ESTERS CEDARWOOD
OIL
LIMONENE
-Similar to xylene
-Does not make
the tissue hard
after prolonged
exposure
-Action is slightly
slower than
xylene
-Flammable and
toxic
-Slower
penetrating
power than
xylene
-Highly volatile,
inflammable and
toxic
-No tissue
shrinkage
-Rarely used now
-Different esters
include amyl
nitrate, methyl
salicylate and
methyl benzoate
-Less toxic, may
be used in manual
processing
-Donot cause
tissue hardening
even after
prolonged
exposure
-Rapid clearing
agent
-Used mainly for
dense tissues
-Clear liquid with
characteristic
“citrus odour”
-No tissue
hardening
-Difficult to
remove from
tissue by paraffin
wax
-Skin sensitizer
and irritant

69. INFILTRATION
 This is the next step after clearing.
 After removing the clearing agent by diffusion,
the tissue space is infiltrated with the embedding
media.
 Most commonly used: molten wax.

70. IDEAL IMPREGNATING MEDIUM
1. Miscible with clearing agent
2. Liquid at higher temperature and solid in room
temperature
3. Homogenous and stable
4. Non toxic and cheap
5. Transparent
6. Fit for tissue sectioning

71. PARAFFIN WAX
 It is a type of hydrocarbon produced as a by
product while refining crude petroleum.
 Most popular universally accepted infiltrating
agent and embedding medium for tissue
processing.
 Non toxic and inexpensive
 Melting point varies between 39°C to 70°C

72.  In the Indian subcontinent, paraffin wax with a
melting point of around 50-60 °C is most suitable
for laboratory use.
 Tissue blocks made in paraffin can be stored for a
long duration
 Causes tissue shrinkage and hardening if
impregnated for too long.

73. Occasions where paraffin wax becomes an
unsuitable media:
1. Processing reagents remove or destroy the tissue
components which are the object of investigation
eg: Lipids
2. When the use of heat may have an adverse effect
on a tissue component eg: enzymes
3. Sections are required to be thinner eg: electron
microscopy
4. Infiltrating media is not sufficiently hard to
support the tissue. Eg: Undecalcified bone

74. RESIN: AGAR: GELATIN CELLOIDIN
Exclusively used for
electron
microscopy, ultra
thin sectioning and
undecalcified bone.
Mainly used as a
cohesive agent for
small, friable tissue
after fixation
(=double
embedding)
-Primarily used for
sectioning of whole
organs
-Used along with
agar as a
preembedded
media and in frozen
sectioning
-Used when
processing dense
and/or hard tissues.
-Use requires
special equipment
and non
conventional
microtomy
techniques.
-Not suitable for
clinical histological
laboratories

75. EMBEDDING(BLOCKING)
 This is the process of creating tissue blocks by
using external support medium to enable
microtomy.
 The embedding media should be compatible with
the infiltrating media to prevent tissue section
separation and to facilitate sectioning.
 Embedding can be done using paraffin wax or
resins.

76. PARAFFIN WAX EMBEDDING
 Most laboratories use modular embedding centres
consisting of a paraffin wax dispenser, cold plate
and heated storage area for molds and processed
tissue cassettes.

77. Paraffin
wax is
dispensed
from
nozzle
into a
warm
mold of
suitable
size
Tissue is
oriented
in the
mold
Tissue is
fixed in
the
bottom
Casette is
placed on
top of the
mold and
filled with
wax
Final
block is
placed on
the cold
plate
Allowed
to solidify

78. TISSUE ORIENTATION
 The ability to see the desired tissue morphology is
based on the orientation of the sample in the
block.
 INCORRECT ORIENTATION: Damages diagnostic
tissue elements during microtomy or obscure
them from microscopic view, preventing correct
diagnosis.

79. TISSUE PROCESSORS
 Tissue processing can be done manually or by
automated processors.
 Manual tissue processing is now done only in small
laboratories.
 Mostly automated tissue processors are currently
in use.
 Automated tissue processors are of two types:
1. Tissue transfer processor
2. Fluid transfer processor

80. MANUAL TISSUE PROCESSOR
 It is rarely used. The advantages are:
1. Small number of samples can be processed.
2. Careful monitoring in each step is possible.
3. Used in case of emergencies or in low resource
settings
4. It is possible to select the reagents of choice with
flexibility in time duration.

81. TISSUE TRANSFER PROCESSOR
Tissues in 30-
100 cassette
in a basket
Different
reagents are
put in
containers
called
carousels
Basket is
submerged in
each carousel
for a specific
time with
gentle
agitation in
each carousel
Basket lifted
up once time
in that
particular
reagent is
complete
Basket
shifted
automatically
to next
carousel

82. HISTOKINETTE AUTOMATED TISSUE
PROCESSORS USED IN OUR INSTITUTE

83.  After grossing, sections are
taken and placed in this
cassette.
 Volume of each cassette is 2
cc.
 No of casettes in each
processor at a time:
Sakura: 28
Leica: 54

84. XYLENE: Kept for 4-6 hours
PARAFFIN WAX:
Kept for 4-6.5
hours
GRADED
ALCOHOL:
Kept for
1.5-2 hours
in each
carousels
TOTAL TIMING:
Ideally 24 hours
but for faster
processing due to
specimen
burden, we keep
it for 16-18
hours.

85. PROCESSING SCHEDULE
Processor: Leica tissue processor
Time: Overnight- 22-24 hours
Tissue: General surgical specimens
STATION AND REAGENT PROCESSING TIME
1- 70% alcohol 2 hours
2- 80% alcohol 2 hours
3- 90% alcohol 2 hours
4- 100% alcohol 2 hours
5- 100% alcohol 2 hours
6- 100% alcohol 2 hours
7- Xylene 1.5 hours
8- Xylene 1.5 hours
9- Xylene 1.5 hours
10- Paraffin wax 2 hours
11- Paraffin wax 2 hours
12- Paraffin wax 2 hours

86. Casettes with tissue after
processing completed
Ready to be embedded!

87. FLUID TRANSFER PROCESSOR
- Completely closed processor.
- In this processor, each step can be customized for
vaccum, temperature and time duration.
Tissue is kept
in container
Container
periodically
filled with
reagent for
first step
Time for
reagent
complete
Fluid
pumped out
of container
Container
filled with
reagent for
the next step

88. MICROWAVE PROCESSING
 Instantaneous heat generation: reduces
processing time
 Heat artifacts can easily be avoided.
 These days “continuous input rapid tissue
processors” are in use, which employ microwave
technology, vaccum infiltration and proprietary
reagents.

89. FUTURE OF FIXATION AND TISSUE
PROCESSING
 In future, there may be environmental friendly
fixatives and fixatives that are more
compatible with molecular techniques and high
resolution microscopy.
 Currently there is ongoing research about
automation of the process of fixation and
integration with genomics.
 Future tissue processing methods may aim to
better preserve molecular information,
including DNA, RNA, and proteins.

90. REFERENCES
Bancroft’s Theory and Practice of Histological Techniques,
Eighth edition, 2019.